The present invention relates to isolated nucleic acids encoding Prolactin Regulatory Element Binding (PREB) protein and recombinant proteins encoded thereby. The nucleic acid sequences are useful in the production of recombinant PREB, as probes, and in the control of prolactin gene expression. In particular embodiments of the invention, PREB nucleic acid sequences are used to detect transcripts of the gene in astrocytomas. The PREB protein is associated with the kinase-mediated hormonal regulation of prolactin gene expression, and may be used as a trans-acting control of transcription.
2.1. FUNCTION OF THE PROLACTIN HORMONE
Prolactin (PRL) is an anterior pituitary hormone that is part of a family of hormones. Prolactin was discovered in 1928 based on the ability of pituitary extracts to cause lactation in pseudo-pregnant rabbits. Cooke, N. E., and Leibhaber, S. A., 1995, Vitamins and Hornones 50:385-459. Accumulated data now suggest a very broad spectrum of roles for PRL. PRL is linked to over three-hundred separate actions in vertebrates including effects on water and salt balance, growth and development, metabolism, brain behavior, reproduction, and immune regulation and protection. Bole-Feysot C. et al.,1998, Endocr. Rev. June; 19(3):225-268. Additionally, a number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL. Bole-Feysot C. et al., 1998, Endocr. Rev. June; 19(3):225-268.
Studies have shown that female transgenic mice over-expressing the rat PRL gene all develop mammary carcinomas at 11-15 months of age and male transgenic mice over-expressing PRL develop dramatic enlargement of the prostate gland. Wennbo H. et al., 1997, J Clin. Invest. December 1; 100 (11):2744-2751. The effect of PRL on cell proliferation was studied in a mouse mammary tumor cell line. The results of the study indicated that PRL antiserum is able to inhibit cell growth by 70% suggesting that PRL may be acting as a local growth factor that stimulates the proliferation of mammary tumors. Mersho, J. et al., 1995, Endocrinology. August; 136(8):3619-3623. Similarly, human breast cancer cells synthesize and secrete biologically active PRL and there is evidence to support that PRL may be involved in an autocrine/paracrine stimulatory loop within breast tissues that may play a role in the pathogenesis of breast cancer. Clevenger C. V. et al., 1995, Am. J Pathol. March; 146(3):695-705.
In addition, PRL is produced by leukocytes and fibroblasts and animal model studies suggest that increased levels of serum PRL may influence the course of arthritis, lupus, and autoimmune type I diabetes, indicating that PRL may play a role in autoimmune diseases and the regulation of immune responses. Neidhart, M., 1998, Proc. Soc. Exp. Biol. Med. April; 217(4):408-419. Ferrag, F. et al., 1997, Cytokines Cell Mol Ther. September; 3(3):197-213. There an important aspect of therapeutic approaches with respect to these diseases is the understanding of the regulation of PRL expression.
2.2. TISSUE SPECIFIC EXPRESSION OF PROLACTIN
The prolactin gene appears to function as an important element in tissue-specific and developmentally regulated gene expression. Cooke, N. E. and Liebhaber, S. A., 1995, Vitamins and Hormones 50:385-459. Prolactin is expressed in a cell-type specific fashion in pituitary lactotropic cells. Cooke, N. E. and Liebhaber, S. A., 1995, Vitamins and Hormones 50:385-459. In addition, prolactin expression has been found in human endometrial cells, human breast tissue, human mammary cell lines, human ovaries, human immune system cells and tissues (thymus, spleen, tonsil, lymph node, lymphocytes, and lymphoid tumors), epithelial cells, vascular endothelial cells, hypothalamic cells, and in human decidua-chorion. Tanaka, S. et al., 1996, Eur. J Endocrinol. 135(2):177-183; Shaw-Bruha, C. M., 1997, Breast Cancer Res. Treat. 44(3):243-253; Schwarzler, P. et al., 1997, Fertil. Steril. 68(4):696-701; Wu, H. et al., 1996, Endocrinology 137(1):349-353; Clapp, C. et al., 1994, Proc. Natl. Acad. Sci. USA 91(22):10384-10388; Clements, J., 1983, Endocrinology 112(3):1133-1134. Among human tissues which do not express prolactin are lung and kidney as well as many others. Schwarzler, P. et al., 1997, Fertil. Steril 68(4):696-701. Although it has been shown that prolactin is expressed in a tissue dependent manner, it is not clear which transcription factors are responsible. While Pit-1, the POU homeo-domain transcription factor (a variant of the helix-turn-helix type transcription factor), gene expression has been shown to follow levels of prolactin expression, Pit-1 is expressed in 3 cell types of the pituitary (thyrotropic cells, lactotropic cells, somoatotropic cells, somatolactortropic cells) while prolactin is primarily expressed in lactotropic cells. Crenshaw, E. B. et al., 1989, Genes Dev. 3(7):959-972. The mechanism of tissue specific expression of prolactin is still being investigated.
2.3. PRL REGULATION
Transcription factors are proteins that bind to regulatory elements in genes and have a critical role in gene regulation and protein expression during development, cellular growth and differentiation. Transcription factors generally can be categorized into four major groups according to the motif in their DNA-binding domains which include (1) the helix-turn-helix group, (2) the zinc finger group, (3) the leucine zipper group, and (4) the helix-loop-helix group. Lloyd, R. V. and Osamura, R. Y., 1997, Microsc. Res. Tech. 3 9(2):168-181.
The prolactin promoter contains multiple binding sites implicated in basal PRL expression and in kinase-mediated hormonal regulation of the gene. The pituitary-specific transcription factor Pit-1 has been shown to play an important role in the expression by the pituitary of the prolactin gene both in development and in the mature organism. Iverson, R. A. et al., 1990, Mol. Endocrinol. 4:1564-1571. Okimura, Y. et al., 1994, Mol. Endocrinol. 8:1559-1565. Although Pit-1 binds to many of the binding sites in the PRL promoter, it does not appear to be responsible for kinase-mediated hormonal regulation of PRL. Fischberg, D. J. et al., 1994, Mol. Endocrinol. 8:1566-1573; Okimura, Y. et al., 1994, Mol. Endocrinol. 8:1559-1565; Howard, P. W. and Maurer, R. A., 1994, J Biol. Chem. 269:28662-28669. This suggests that there are factors other than Pit-1 that are responsible for the regulation of PRL gene expression.
Oct-1 and TEF are two other transcription factors which can bind to the promoter of the PRL gene. Voss, J. W. et al., 1991, Genes Dev. 5:1309-1320; Drolet, D. W. et al., 1991, Genes Dev. 5:1739-1753. Studies show that Oct-1 is unlikely to be involved in the kinase-mediated transcription of the prolactin gene since protein kinase A (PKA)-mediated phosphorylation of Oct-1 decreases its DNA binding activity. Segil, N. et al., 1991, Science 254:1814-1816. Additionally, TEF is not likely to be involved in the kinase-mediated transcription of the prolactin gene since its mode of action is apparently limited to thyrotroph development. Voss, J. W. et al., 1991, Genes Dev. 5:1309-1320.
Mutations in the Pit1 gene, observed in the naturally occurring Snell (dw) and Jackson (dwJ) mutant mice, result in a murine phenotype of severe growth retardation and a remarkably hypoplastic pituitary gland, due to a developmental failure of the three anterior pituitary cell types that specifically express the hormones regulated by Pit1. See Li et al., Nature 347:528-533 (1990). Mutations in the human homologue of Pit1 have also been shown to be responsible for deficiencies of these three pituitary hormones, and to result in a human phenotype of growth abnormalities, severe mental retardation, failure of lactation, congenital hypoparathyroidism, facial dysmorphism and hypoplastic pituitary. See Radovick et al, Science 57:1115-1118 (1992); Tatsumi et al., Nat. Genet. 1:56-58 (1992); de Zegher et al, J Clin. Endocr. Metab. 80:3127-3130 (1995).
Despite evidence that PRL gene expression is highly regulated and induced by the cAMP-protein kinase-A pathway, a transcription factor involved in the kinase-mediated transcription of the prolactin gene has been, prior to the present invention, elusive. Keech, C. A. et al., 1992, Mol Endocrinol 6(12):2059-2070. Evidence suggests that there is a ubiquitous transcription factor that is involved in the PKA-mediated transcriptional activation pathway of prolactin. Rajnarayan, S. et al., 1995, Mol Endocrinol 9(4):502-512. A better understanding of the PKA-mediated transcriptional activation pathway, coupled to more effective means to control this pathway, could lead to the treatment of ailments that are related to the inappropriate expression of the prolactin gene.
2.4TREATMENT OF DISEASE THROUGH PROLACTIN REGULATION
Since its discovery in 1928, PRL has been implicated in a broad spectrum of roles including regulation of reproductive function, control of metabolism, osmoregulation, and immune regulation, as well as growth and development in certain vertebrate species. Nicoll, C. S., 1991, Perspect. Biol. Med. 25:369-381. In addition to becoming well known as an important regulator of immune function, a number of disease states, including different forms of cancer, autoimmune diseases, developmental diseases and osteoporosis have been connected to the overproduction of PRL. Yu-Lee, L-y., 1997, Proc. Soc. Exp. Biol. Med. 215:35-52; Adler et al., Metabolism, 47:425-428 (1998).
PRL expression has been detected in human mammary tumors, and human mammary tumor cell lines can produce PRL, indicating a possible auto/paracrine function of PRL in mammary tumor growth. Wennbo, H. et al., 1997, J. Clin. Invest. 100:2744-2751. In addition, inhibitors of the proliferation of human breast cancer cells in vitro appear to inhibit endogenous prolactin action at the level of the prolactin receptor and antibodies to PRL inhibit the proliferation of rat mammary tumor cells in vitro. De Petrocellis, L. et al., 1998, Proc Natl. Acad. Sci. USA 95(14):8375-8380; Mersho, J. et al., 1995, Endocrinology 136(8):3619-3623. Furthermore, tamoxifen, an anti-estrogen which is known for its anti-tumoral action in vivo, inhibits PRL-induced activation of kinases as well as PRL binding and cell growth indicating the possible role of PRL inhibition in the treatment of breast cancer. Das, R. and Vonderhaar, B. K., 1997, Cancer Letters 116(1):41-46. Another study aimed at further characterizing the role of prolactin in breast cancer has focused on the creation of transgenic mice that over-express prolactin. Significantly, all of the female mice which over-expressed PRL developed mammary carcinomas at 11-15 months of age. Wennbo, H. et al., 1997, J. Clin. Invest. 100:2744-2751. In the same study, organ culture experiments were conducted which demonstrated an autocrine/paracrine effect of PRL.
The role of PRL in human breast cancer has been suggested in a study that reports two cases of breast cancer associated with prolactinoma. Strungs, I. et al., 1997, Pathology 29(3):320-323. Correspondingly, male mice which over-expressed PRL developed dramatic enlargement of the prostate gland, approximately 20 times the normal size. Wennbo, H. et al., 1997, Endocrinology 138(10)4410-4415. It is interesting to note that the level of PRL increases with age, coinciding with development of prostate hyperplasia in humans. Hammond, G. L. et al., 1997, Clin. Endocrinol. 7:129-135. It appears therefore that PRL may be an important factor in the etiology of prostate diseases including cancer. All the aforementioned studies are in strong support of the notion that PRL may be an important target in the quest to control cancer.
PRL has also been implicated in autoimmune diseases. PRL, as well as growth hormone, is required for the development of mature lymphocytes and for the maintenance of immunocompetence. Berczi, I., 1997, Acta Paediatr Suppl. 423:70-75. It is suggested that hyperprolactinemia is a risk factor for the development of autoimmunity. There are many studies that support a role of PRL in modulating the immune response and altered PRL levels have been observed in animal models of autoimmune diseases such as lupus erythematosus, diabetes, rheumatoid arthritis, and collagen type II-induced arthritis. Berczi, I., 1983, et al. Acta. Endocrinol. 102:351-357; Jara, L. J. et al., 1992, Am. J. Med. Sci. 303:222-226; Neidhart, M., 1998, Proc. Soc. Exp. Biol. Med. 217(4):408-419. Since PRL constitutes a stimulatory link between the neuroendocrine and immune systems, increased serum levels may activate a hyperimmune response. Neidhart, M., 1998, Proc. Soc. Exp. Biol. Med. 217(4):408-419. In these diseases, it may be advantageous to reduce the serum levels of PRL by inhibiting its gene expression. Additionally, serum PRL levels were elevated in human bone transplantation patients which exhibited chronic graft-versus-host disease indicating that prolactin is a mediator of graft-versus-host disease. Hinterberger-Fischer, M. et al., 1994, Bone Marrow Transplant 14(3):403-406. The inhibition of PRL after transplantation may decrease the rejection problems of transplantation patients.
In contrast, an increase in prolactin expression may be beneficial where the immune response is compromised, for example, in AIDS. Recombinant human PRL has been demonstrated to have the ability to stimulate proliferation of B-cell hybridomas in a dose-dependent manner, resulting in an overall increase of antibody production. Richards, S. M. et al., 1998, Cell. Immunol. 184(2):85-91. Additionally, PRL was able to overcome the growth inhibition of the hybridoma cells by transforming growth factor beta (TGF-beta), indicating a role for PRL in the treatment of diseases associated with over-expression of TGF-beta, or in AIDS where it may be advantageous to stimulate lymphoid cells. Hinterberger-Fischer, M. et al., 1994, Bone Marrow Transplant 14(3):403-406.
PRL may regulate bone marrow function as well. In rats which have their pituitary gland removed, treatments with PRL reversed the anemia, leucopenia, and thrombocytopenia in their bone marrow. Nagy, E. and Berczi, I., 1989, Br. J. Haematol. 71(4):457-462. Moreover, prolactin regulation of the growth of hematopoetic progenitors in a bone marrow stroma environment has been demonstrated in vitro by the addition of PRL antibodies to cultures resulting in a reduction of hematopoetic progenitor colonies. Bellone, G., 1997, et al. Blood 90(1):21-27. These findings suggest that over-expression of prolactin may be used to stimulate the growth of hematopoetic progenitor cells in vitro which may ultimately be transplanted into a patient.
Studies have also linked PRL overproduction with osteoporosis. Humans with prolactinoma are at risk for reproductive disorders and osteoporosis which may be due to PRL-induced hypogonadism. See Adler et al., Metabolism 47:425-428 (1998). In addition, anovulation condition (which is an estrogen deficiency due to high prolactin levels) is linked with premature bone mass loss. See Koloszar et al., Orv. Hetil. 138:71-73 (1997). It has been suggested that osteopathy in hyperprolactinemic hypogonadism is due to reduced bone formation and not reduced estradiol production, indicating a link of PRL levels with bone formation. See Rozhinskaia et al., Probl. Endokrinol. 38:17-19 (1992). Similarly, Ciccarelli et al. (Clin. Enndocrinol. 28:1-6 (1988)) have suggested that there is a direct effect of PRL on bone mass. The risk of developing osteoporosis due to increased levels of PRL has been seen in women as well as in men. See Jackson et al., Ann Intern. Med. 105:543-545 (1986).
The present invention relates to the discovery of a novel transcription factor, called PREB (Prolactin Regulatory Element Binding) protein which functions in the kinase-mediated hormonal regulation of prolactin gene expression. The invention is based, at least in part, on the discovery and characterization of the rat Preb gene and protein and the identification of a cloned human DNA containing the human PREB gene.
In a first series of embodiments, the present invention provides for a nucleic acid molecule encoding PREB, and a PREB protein having an amino acid sequence as encoded by that nucleic acid which binds to the 1P element of the PRL promoter.
In a second set of embodiments, the present invention provides for a method of inhibiting kinase-mediated transactivation of prolactin gene expression.
In a third set of embodiments, the present invention provides for an assay for distinguishing between different brain tumor types whereby PREB transcript levels are quantified. For example, but not by way of limitation, PREB transcript levels can be quantified using a PREB nucleic acid sequence as a probe, by RT-PCR analysis or by microarray analysis. The presence of PREB would indicate the presence of astrocytoma brain tumor cells but not neuroepithelioma or glioma brain tumor cells.
In a fourth set of embodiments, the present invention provides for methods for the treatment of cancers and autoimmune diseases through the inhibition of prolactin gene expression.
In a fifth set of embodiments, the present invention provides for methods for improving the immune response by increasing prolactin gene expression.
In a sixth set of embodiments, the present invention provides for a method for inhibiting Pit-1-mediated transactivation of prolactin gene expression and the transactivation of other genes whose transcriptional activation is controlled by Pit-1.
In a seventh set of embodiments, the present invention provides for methods for stimulating the growth of cells both in vitro and in vivo through the over-expression of genes by the use of promoter sequences which are regulated by the binding of the PREB transcription factor. This method is also useful in the culture of skin cells or bone marrow cells in vitro which will ultimately be transplanted into a patient.
In an eighth set of embodiments, the present invention provides for a method for inhibiting graft-versus-host disease in transplant patients through the inhibition of prolactin gene expression.
In a ninth set of embodiments, the present invention provides for a method for controlling development through the inhibition of PREB expression.
In a tenth set of embodiments, the present invention provides for a nucleic acid encoding human PREB, as contained in plasmid pCRScript (Stratagene) deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Va. 20110-2209 USA) and assigned accession number PTA1259. The present invention also provides for two genomic fragments, which encompass the PREB gene minus 500 bp from the 3xe2x80x2 UTR that is within the cDNA transcript, of 1.6 kb and 2.5 kb respectively, which were deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Va. 20110-2209 USA) and assigned accession numbers PTA1258 and PT01260 respectively.
In an eleventh set of embodiments, the present invention provides for a method of detecting trisomy 2p whereby a PREB nucleic acid sequence is used as a probe. The presence of additional copies of PREB would indicate a trisomy 2p condition.
In a twelfth set of embodiments, the present invention provides for a method of detecting an increased propensity to develop osteoporosis whereby any change in the expression of PREB which results in activation or inactivation of the PREB protein would indicate a propensity to develop osteoporosis. For example, changes which could indicate an increased propensity to develop osteoporosis include, but are not limited to, (1) changes in the PREB gene, such as heterozygous or homozygous partial or total deletions of the PREB gene, insertions, base pair changes, mutations, etc.; (2) changes in the transcript levels; and/or (3) changes in the PREB protein levels and truncations of the PREB protein (which may be the result of a mutation in the PREB gene) which could result in an up-regulation of certain pathways.
In a thirteenth set of embodiments, the present invention provides for a method of treating osteoporosis, or lowering the likelihood of developing osteoporosis comprising administering the PREB gene or gene product, including, but not limited to antisense PREB mRNA, or the PREB protein, to a subject.